**4. Absolute quantification of prospective biomarkers in blood plasma with isotopically labeled internal standards**

Absolute quantification of glycation rates at individual modification sites by means of isotopically labeled standards was for the first time proposed by Zhang and co-workers in 2013 [86]. For the early diagnosis of T2DM glycation at the most abundant human plasma protein HSA was monitored by quantitative analysis of its characteristic tryptic peptides. Thereby, all probands were classified into three groups: T2DM (n = 73), IGT (n = 63), and NGT (n = 253). In this study 18O-labeling was used to screen glucose-sensitive and glucose-insensitive peptides within HSAderived peptides. Glucose-sensitive peptides tested as biomarker candidates for T2DM in a clinical plasma samples, and glucose-insensitive peptide was selected as the internal standards. Three peptides (LDELRDEGK (K190), FKDLGEENFK (K12), and KVPQVSTPTLVEVSR (K414)) showed significant different in their concentrations in the T2DM group compared with the IGT group. Among them two peptides FKDLGEENFK and KVPQVSTPTLVEVSR exhibited significant differences between both NGT/IGTand IGT/T2DM groups indicating that these peptides could be used as potential biomarkers for the early diagnosis of T2DM. It is important to mention that peptides FKDLGEENFK and KVPQVSTPTLVEVSR showed excellent sensitivities (97.23 and 94.47%, respectively) and specificities (93.65 and 98.41%, respectively) between the NGT/IGT groups. For the NGT/IGT and T2DM groups sensitivity of peptides FKDLGEENFK and KVPQVSTPTLVEVSR was 97.06 and 99.27%, respectively, and specificities was 97.23 and 94.47%. It indicates that these peptides could be prospective biomarkers for the early diagnosis of T2DM [86].

The internal standardization with stable isotope-labeled synthetic peptides and related stable isotope dilution techniques are widely used in glycation research [84, 95, 111, 112]. A stable isotope label can be easily introduced in the step of amino acid building block during the synthesis of Amadori-modified peptides [115]. These peptides can be directly used in the stable isotope dilution approach for absolute quantification (AQUA) [116]. Implementation of synthetic Amadori-modified peptides increases robustness, precision and accuracy of biomarkers analysis [117].

In the classical way this strategy was established for glycated peptides by Spiller and co-workers [118], who applied six standard synthetic peptides containing two isotope-labeled residues (introduced by Fmoc-(13C6, 15N) leucine and Fmoc- ( 13C5, 15N) proline) along with Amadori modification, introduced by the global post-synthetic glycation approach [119]. The peptides represented six glycation sites in HSA – TCVADESAENCDKSLHTLFGDK (K64), SLHTLFGDKLCTVATLR (K73), AACLLPKLDELRDEGK (K181), ADLAKYICENQDSISSK (K262), VFDEFKPLVEEPQNLIK (K378) and KLVAASQAALGL (K574). The standard mixtures were spiked (25 μL, 1.2 μmol/L) to the tryptic digest, obtained from five T2DM patients and five non-diabetic individuals and, after BAC enrichment and SPE, analyzed by a hybrid quadrupole-linear ion trap MS in the MRM mode with three specific Q1/Q3 *m/z* ranges (transitions) for each analyte [118].

**77**

*Individual Glycation Sites as Biomarkers of Type 2 Diabetes Mellitus*

All six analytes and corresponding standard peptides could be detected in all ten plasma samples. The quantities varied from 22.6 ± 3.3 to 180.3 ± 8.8 pmol per mg plasma protein. Also it was shown that content of all six glycated peptides were statistically different between the two cohorts. This study demonstrated the applicability of the AQUA technique to quantification of glycation sites in plasma proteins. In agreement with the results of LFQ studies [83], individual HSA glycation sites responded to hyperglycaemia in different ways. Interestingly, significantly higher, in comparison to others, abundance of glycated sites K64 and K378 was observed [118]. For absolute quantification of glycated peptides by the standard isotope dilution technique Prof. Stefanowicz and co-workers used bi-labeled peptides that contained

C6

KKDabK represented the sequence of Ig kappa chain C region protein,

tion of individual glycation sites in blood plasma proteins [120].

Later Spiller *et al.* [97] studied the glycation degrees of 27 glycation sites representing nine plasma proteins in 48 newly diagnosed male T2DM patients and 48 non-diabetic individuals. After protein digestion with trypsin, samples were spiked with concentration-balanced mixture of synthetic 13C,15N-labeled glycated peptides (synthesized according Spiller at al [118]) as internal standards. The quantification was based on MRM using specific transitions for each targeted peptide and isotopelabeled internal peptide standards. The samples from two groups of participants were evaluated by different statistical tests (Kolmogorow-Smirnow, Mann–Whitney, and *t* test), classified by a decision tree algorithm using HbA1c in combination with each glycated peptide. Also to find the best feature set for classification, support vector machine-recursive feature elimination (SVM-RFE) method was performed for all glycated peptides and clinical parameters of participants, including HbA1c, fasting plasma glucose (FPG), body mass index (BMI), etc. The most interesting results were obtained for glycated peptide AVGDKLPECEAVCGKPK (K141) from haptoglobin which half-life time is 2–4 days. The combination of two biomarkers of T2DM glycated K141 of haptoglobin and HbA1cprovided a sensitivity of 94%, a specificity of 98%, and an accuracy of 96% to identify T2DM.But a set of 15 features considering three glycation sites in HSA, K141 in haptoglobin, and 11 routine laboratory measures of T2DM, metabolic syndrome, obesity, inflammation, and insulin

KKDabK corresponded to the HSA sequence were synthesized. The mixture of standard stable isotope-labeled peptides (20 pmol each) was added to aliquots of plasma samples (of 5 T2DM patients and 5 non-diabetic individuals). After tryptic digestion, samples were analyzed by QqTOF-MS. Glycated peptides from plasma samples and standards were annotated in MS scans (*m/z* 400–2000) by their tR, *m/z* and isotopic patterns and confirmed by MS/MS analysis using Orbitrap Elite MS. The corresponding MS profiles clearly represented isotopic patterns of both light and heavy peptides with good sensitivities that gave access to reliable quantification by peak areas of analytes and corresponding internal standards. All three potential biomarker peptides demonstrated a significantly higher (in 1.5–1.9-fold) content in diabetic patients, in comparison to that in non-diabetic controls. The obtained results were cross-validated by label-free quantification performed in an independent RP-HPLC-ESI-QqTOF-MS study [120]. The resulting fold changes were close to those observed with the stable isotope dilution approach, proposed by Spiller et al. [118]. It indicates similar power of the both methods for absolute quantifica-

that cleaves during digestion procedure [115]. Later on we have shown applicability of these bi-labeled glycated peptides for the absolute quantification of individual glycation sites in plasma proteins [120]. Based on the previous label-free quantification [83] the prevalent glycated peptides with biomarker properties were chosen. The Amadori-modified standard peptides DSTYSLSSTLTLSKAmADYE13C6

15N2-lysine, and comprise a dabsyl moiety

15N2-KKDabK and VFDEFKAmPLVEEPQNLI13C6

15N2-

15N2-

*DOI: http://dx.doi.org/10.5772/intechopen.95532*

stable isotope label, introduced as 3

ADLAKAmYICENQDSISS13C6

#### *Individual Glycation Sites as Biomarkers of Type 2 Diabetes Mellitus DOI: http://dx.doi.org/10.5772/intechopen.95532*

*Type 2 Diabetes - From Pathophysiology to Cyber Systems*

**with isotopically labeled internal standards**

assess the predictive potential of these markers. However, on this way, the limitations of label-free approach become critical. Indeed, LFQ is disadvantages for analysis of large cohorts due to sensitivity of electrospray ionization technique (ESI) to multiple factors, which is manifested as matrix effects [113]. Thus, the analysis conditions (e.g. temperature, experimenter, column condition) must be as constant as possible, that is difficult to achieve with big batch sizes. And duration of batch analysis can be rather long, as prolonged gradients are often used to improve peptide separation [114]. To overcome these limitations, absolute quantification strategies can be employed [86].

**4. Absolute quantification of prospective biomarkers in blood plasma** 

Absolute quantification of glycation rates at individual modification sites by means of isotopically labeled standards was for the first time proposed by Zhang and co-workers in 2013 [86]. For the early diagnosis of T2DM glycation at the most abundant human plasma protein HSA was monitored by quantitative analysis of its characteristic tryptic peptides. Thereby, all probands were classified into three groups: T2DM (n = 73), IGT (n = 63), and NGT (n = 253). In this study 18O-labeling was used to screen glucose-sensitive and glucose-insensitive peptides within HSAderived peptides. Glucose-sensitive peptides tested as biomarker candidates for T2DM in a clinical plasma samples, and glucose-insensitive peptide was selected as the internal standards. Three peptides (LDELRDEGK (K190), FKDLGEENFK (K12), and KVPQVSTPTLVEVSR (K414)) showed significant different in their concentrations in the T2DM group compared with the IGT group. Among them two peptides FKDLGEENFK and KVPQVSTPTLVEVSR exhibited significant differences

between both NGT/IGTand IGT/T2DM groups indicating that these peptides could be used as potential biomarkers for the early diagnosis of T2DM. It is important to mention that peptides FKDLGEENFK and KVPQVSTPTLVEVSR showed excellent sensitivities (97.23 and 94.47%, respectively) and specificities (93.65 and 98.41%, respectively) between the NGT/IGT groups. For the NGT/IGT and T2DM groups sensitivity of peptides FKDLGEENFK and KVPQVSTPTLVEVSR was 97.06 and 99.27%, respectively, and specificities was 97.23 and 94.47%. It indicates that these peptides could be prospective biomarkers for the early diagnosis of T2DM [86]. The internal standardization with stable isotope-labeled synthetic peptides and

related stable isotope dilution techniques are widely used in glycation research [84, 95, 111, 112]. A stable isotope label can be easily introduced in the step of amino acid building block during the synthesis of Amadori-modified peptides [115]. These peptides can be directly used in the stable isotope dilution approach for absolute quantification (AQUA) [116]. Implementation of synthetic Amadori-modified peptides increases robustness, precision and accuracy of biomarkers analysis [117]. In the classical way this strategy was established for glycated peptides by Spiller

and co-workers [118], who applied six standard synthetic peptides containing

(K73), AACLLPKLDELRDEGK (K181), ADLAKYICENQDSISSK (K262),

three specific Q1/Q3 *m/z* ranges (transitions) for each analyte [118].

15N) proline) along with Amadori modification, introduced by the global post-synthetic glycation approach [119]. The peptides represented six glycation sites in HSA – TCVADESAENCDKSLHTLFGDK (K64), SLHTLFGDKLCTVATLR

VFDEFKPLVEEPQNLIK (K378) and KLVAASQAALGL (K574). The standard mixtures were spiked (25 μL, 1.2 μmol/L) to the tryptic digest, obtained from five T2DM patients and five non-diabetic individuals and, after BAC enrichment and SPE, analyzed by a hybrid quadrupole-linear ion trap MS in the MRM mode with

15N) leucine and Fmoc-

two isotope-labeled residues (introduced by Fmoc-(13C6,

**76**

( 13C5,

All six analytes and corresponding standard peptides could be detected in all ten plasma samples. The quantities varied from 22.6 ± 3.3 to 180.3 ± 8.8 pmol per mg plasma protein. Also it was shown that content of all six glycated peptides were statistically different between the two cohorts. This study demonstrated the applicability of the AQUA technique to quantification of glycation sites in plasma proteins. In agreement with the results of LFQ studies [83], individual HSA glycation sites responded to hyperglycaemia in different ways. Interestingly, significantly higher, in comparison to others, abundance of glycated sites K64 and K378 was observed [118].

For absolute quantification of glycated peptides by the standard isotope dilution technique Prof. Stefanowicz and co-workers used bi-labeled peptides that contained stable isotope label, introduced as 3 C6 15N2-lysine, and comprise a dabsyl moiety that cleaves during digestion procedure [115]. Later on we have shown applicability of these bi-labeled glycated peptides for the absolute quantification of individual glycation sites in plasma proteins [120]. Based on the previous label-free quantification [83] the prevalent glycated peptides with biomarker properties were chosen. The Amadori-modified standard peptides DSTYSLSSTLTLSKAmADYE13C6 15N2- KKDabK represented the sequence of Ig kappa chain C region protein, ADLAKAmYICENQDSISS13C6 15N2-KKDabK and VFDEFKAmPLVEEPQNLI13C6 15N2- KKDabK corresponded to the HSA sequence were synthesized. The mixture of standard stable isotope-labeled peptides (20 pmol each) was added to aliquots of plasma samples (of 5 T2DM patients and 5 non-diabetic individuals). After tryptic digestion, samples were analyzed by QqTOF-MS. Glycated peptides from plasma samples and standards were annotated in MS scans (*m/z* 400–2000) by their tR, *m/z* and isotopic patterns and confirmed by MS/MS analysis using Orbitrap Elite MS. The corresponding MS profiles clearly represented isotopic patterns of both light and heavy peptides with good sensitivities that gave access to reliable quantification by peak areas of analytes and corresponding internal standards. All three potential biomarker peptides demonstrated a significantly higher (in 1.5–1.9-fold) content in diabetic patients, in comparison to that in non-diabetic controls. The obtained results were cross-validated by label-free quantification performed in an independent RP-HPLC-ESI-QqTOF-MS study [120]. The resulting fold changes were close to those observed with the stable isotope dilution approach, proposed by Spiller et al. [118]. It indicates similar power of the both methods for absolute quantification of individual glycation sites in blood plasma proteins [120].

Later Spiller *et al.* [97] studied the glycation degrees of 27 glycation sites representing nine plasma proteins in 48 newly diagnosed male T2DM patients and 48 non-diabetic individuals. After protein digestion with trypsin, samples were spiked with concentration-balanced mixture of synthetic 13C,15N-labeled glycated peptides (synthesized according Spiller at al [118]) as internal standards. The quantification was based on MRM using specific transitions for each targeted peptide and isotopelabeled internal peptide standards. The samples from two groups of participants were evaluated by different statistical tests (Kolmogorow-Smirnow, Mann–Whitney, and *t* test), classified by a decision tree algorithm using HbA1c in combination with each glycated peptide. Also to find the best feature set for classification, support vector machine-recursive feature elimination (SVM-RFE) method was performed for all glycated peptides and clinical parameters of participants, including HbA1c, fasting plasma glucose (FPG), body mass index (BMI), etc. The most interesting results were obtained for glycated peptide AVGDKLPECEAVCGKPK (K141) from haptoglobin which half-life time is 2–4 days. The combination of two biomarkers of T2DM glycated K141 of haptoglobin and HbA1cprovided a sensitivity of 94%, a specificity of 98%, and an accuracy of 96% to identify T2DM.But a set of 15 features considering three glycation sites in HSA, K141 in haptoglobin, and 11 routine laboratory measures of T2DM, metabolic syndrome, obesity, inflammation, and insulin

resistance provided a sensitivity of 98%, a specificity of 100%, and an accuracy of 99% for newly diagnosed T2DM patients. This study shows great potential of glycation sites in plasma proteins providing an additional diagnostic tool for T2DM and elucidating that the combination of these sites with HbA1c and FPG could improve the diagnosis of T2DM. The combination of both biomarkers HbA1c and glycated haptoglobin with half-life times 2 to 4 days is sensitive to long- and short-term fluctuations of blood glucose concentrations [97].

Selected 27 glycated peptides were tested further in the quality of multiple biomarker set [121]. For this plasma samples from 48 patients with duration of T2DM for more than 10 years, 48 non-diabetic individuals and 20 pre-diabetic persons we examined. The strategy of analysis was the same as described above [97]. In longterm controlled T2DM patients, 27 glycated peptides were detected at significantly higher levels and provided moderate diagnostic accuracies (ACCs) from 61 to 79%, resulting in sub-grouping of patients in three distinct clusters. In this study, a feature set of one glycated peptides from haptoglobin (K141) and 6 established clinical parameters provided an ACC of 95%. The same number of clusters was identified in pre-diabetic males (ACC of 95%) using a set of 8 glycation sites (mostly from HSA). Re-examination of all patients present in one cluster showed progression of pre-diabetic state or advanced towards diabetes in the following five years. Overall, the studied glycation sites can play a role of promising biomarkers for sub-grouping pre-diabetic patients to estimate their risk for the development of T2DM [121].

Together with our recent report [84] these work clearly indicate under-explored potential of integrated peptide biomarkers. Moreover, it is well seen, that absolute quantification approaches are preferable, due to their higher precision and lower method-related dispersion. Thus, it is obvious, that after the explorative LFQ-based study, follow-up absolute quantification screening of large cohorts is necessary.

Besides 18O-labeling and AQUA, tag-based labeling approaches were used for quantification of glycation sites. Thus, Qiu*et al*. proposed the use of isobaric tags for relative and absolute quantification (iTRAQ ) to reveal differences in HSA glycation patterns between healthy individuals and diabetic patients [88]. The authors described *in vitro* and *in vivo* experiments have been carried out to evaluate the impact of HSA glycation on the binding to anticoagulant drugs (warfarin and heparin). Plasma samples from 32 diabetic patients and 33 healthy individuals were treated with polyethylene glycol for precipitation of immunoglobulins. After this, the glycated albumin (GA) was separated using BAC. Trypsin digested GA was separated on C18-AQ analytical column and analyzed using a LTQ-Orbitrap Velos Pro MS operated in DDA mode with CID and further with ETD fragmentation. For quantitative analysis, the digested samples were labeled with iTRAQ 8-plex reagents. After labeling, the 8 samples with different mass tags were evenly mixed and glycated peptides with iTRAQ labels were extracted from the pooled samples using BAC. MS analysis was performed on a LTQ-Orbitrap Fusion MS operated in the DDA mode for CID-MS/MS and HCD-MS3 . A total of 49 glycation sites (including 43 glycated lysines and six glycated arginines) on GA were successfully identified using this approach. Among them seven glycation sites, R81, R117, R186, R257, K313, R410 and K541, were discovered for the first time. It is interesting that glycation at sites K4, R81, R117, K439, K519, K538, K541, K557 and K573 were specifically present in diabetic patients, while two sites, R410 and K436, were found only in healthy subjects. Altogether 21 glycation sites were quantified, and 19 of them, including K51, K64, K93, K162, K199, K233, K262, K313, K323, K378, K402, K414, K466, K475, K525, K545, K557, K564 and K574, showed statistically enhanced glycation during diabetes [88].

Examples of methods used for absolute quantification of prospective biomarkers in blood plasma with isotopically labeled internal standards are summarized in **Table 2**.

**79**

**#**

1 2 3 4 5 6 **Table 2.**

*(spec.)*

HSA (K51, K64, K93, K162, K199, K233, K262, K313, K323, K378,

K402,K414, K466, K475, K525, K545, K557, K564 and K574)

Haptoglobin (K141), HSA (K262, K378, K73, K525, K574, K359, K174,

K64), serotransferrin (K683)

Haptoglobin (K141), HSA (K93, K262, and K414)

13C,15N-labelling

13C,15N-labelling

18O-labeling *Overview of methods used for absolute quantification of prospective biomarkers in blood plasma with isotopically labeled internal standards. Accuracy (accur.), sensitivity (sens.), specificity* 

ESI-QqLIT ESI-QqLIT

ESI-LTQ-Orbitrap

Statistical difference between control and

[88]

T2DM p < 0.05

Ig kappa chain C region protein (K75), HSA (K286, K402)

*L*-lysine-3

C615N2 and dabsyl moiety

ESI-QqTOF

Significantly higher

[120]

(in 1.5–1.9-fold)

content in T2DM

Sens. 98%, spec.

[97]

100%, accur. 99%

Diagnostic accuracy

[121]

95%

HSA (K64, K73, K181, K262, K378, K574).

HSA (K414)

**Proteins and sites of glycation**

**Peptide labeling**

18O-labeling *L*-leucine-13C6,15N, *L*-proline 13C5,15N

ESI-QqLIT

Statistical difference

[118]

between control and

T2DM: p < 0.0001 or

p < 0.05

**MS** ESI-QqTOF

**Method power**

Sens. 99.27%, spec.

[86]

94.47%

**Ref**

*Individual Glycation Sites as Biomarkers of Type 2 Diabetes Mellitus*

*DOI: http://dx.doi.org/10.5772/intechopen.95532*


#### *Individual Glycation Sites as Biomarkers of Type 2 Diabetes Mellitus DOI: http://dx.doi.org/10.5772/intechopen.95532*

*Type 2 Diabetes - From Pathophysiology to Cyber Systems*

fluctuations of blood glucose concentrations [97].

the DDA mode for CID-MS/MS and HCD-MS3

resistance provided a sensitivity of 98%, a specificity of 100%, and an accuracy of 99% for newly diagnosed T2DM patients. This study shows great potential of glycation sites in plasma proteins providing an additional diagnostic tool for T2DM and elucidating that the combination of these sites with HbA1c and FPG could improve the diagnosis of T2DM. The combination of both biomarkers HbA1c and glycated haptoglobin with half-life times 2 to 4 days is sensitive to long- and short-term

Selected 27 glycated peptides were tested further in the quality of multiple biomarker set [121]. For this plasma samples from 48 patients with duration of T2DM for more than 10 years, 48 non-diabetic individuals and 20 pre-diabetic persons we examined. The strategy of analysis was the same as described above [97]. In longterm controlled T2DM patients, 27 glycated peptides were detected at significantly higher levels and provided moderate diagnostic accuracies (ACCs) from 61 to 79%, resulting in sub-grouping of patients in three distinct clusters. In this study, a feature set of one glycated peptides from haptoglobin (K141) and 6 established clinical parameters provided an ACC of 95%. The same number of clusters was identified in pre-diabetic males (ACC of 95%) using a set of 8 glycation sites (mostly from HSA). Re-examination of all patients present in one cluster showed progression of pre-diabetic state or advanced towards diabetes in the following five years. Overall, the studied glycation sites can play a role of promising biomarkers for sub-grouping pre-diabetic patients to estimate their risk for the development of T2DM [121].

Together with our recent report [84] these work clearly indicate under-explored potential of integrated peptide biomarkers. Moreover, it is well seen, that absolute quantification approaches are preferable, due to their higher precision and lower method-related dispersion. Thus, it is obvious, that after the explorative LFQ-based study, follow-up absolute quantification screening of large cohorts is necessary. Besides 18O-labeling and AQUA, tag-based labeling approaches were used for quantification of glycation sites. Thus, Qiu*et al*. proposed the use of isobaric tags for relative and absolute quantification (iTRAQ ) to reveal differences in HSA glycation patterns between healthy individuals and diabetic patients [88]. The authors described *in vitro* and *in vivo* experiments have been carried out to evaluate the impact of HSA glycation on the binding to anticoagulant drugs (warfarin and heparin). Plasma samples from 32 diabetic patients and 33 healthy individuals were treated with polyethylene glycol for precipitation of immunoglobulins. After this, the glycated albumin (GA) was separated using BAC. Trypsin digested GA was separated on C18-AQ analytical column and analyzed using a LTQ-Orbitrap Velos Pro MS operated in DDA mode with CID and further with ETD fragmentation. For quantitative analysis, the digested samples were labeled with iTRAQ 8-plex reagents. After labeling, the 8 samples with different mass tags were evenly mixed and glycated peptides with iTRAQ labels were extracted from the pooled samples using BAC. MS analysis was performed on a LTQ-Orbitrap Fusion MS operated in

ing 43 glycated lysines and six glycated arginines) on GA were successfully identified using this approach. Among them seven glycation sites, R81, R117, R186, R257, K313, R410 and K541, were discovered for the first time. It is interesting that glycation at sites K4, R81, R117, K439, K519, K538, K541, K557 and K573 were specifically present in diabetic patients, while two sites, R410 and K436, were found only in healthy subjects. Altogether 21 glycation sites were quantified, and 19 of them, including K51, K64, K93, K162, K199, K233, K262, K313, K323, K378, K402, K414, K466, K475, K525, K545, K557, K564 and

Examples of methods used for absolute quantification of prospective biomarkers in blood plasma with isotopically labeled internal standards are summarized in

K574, showed statistically enhanced glycation during diabetes [88].

. A total of 49 glycation sites (includ-

**78**

**Table 2**.

*Overview of methods used for absolute quantification of prospective biomarkers in blood plasma with isotopically labeled internal standards. Accuracy (accur.), sensitivity (sens.), specificity (spec.)*
